• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH    ₂ (Catalog # ). The specific activity is >8,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Ala16-Ser247, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala16

• Structure / Form

  Pro form

• Predicted Molecular Mass

  26 kDa

• SDS-PAGE

  34 kDa, reducing conditions

Assay Procedure

Materials

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)

• Assay Buffer: 0.1 M Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 (w/v), pH 8.0

• Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # )

• Recombinant Human Trypsin 2/PRSS2 (rhTrypsin 2) (Catalog # 3586-SE)

• Bacterial Thermolysin (Thermolysin) (Catalog # )

• 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO

• Substrate II: MCA-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ), 2 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Activate rhEnterokinase with Thermolysin.

1. Dilute rhEnterokinase to 100 µg/mL in Activation Buffer.

2. Dilute Thermolysin to 3.16 µg/mL in Activation Buffer.

3. Mix 50 µL of 100 µg/mL rhEnterokinase with 50 µL of 3.16 µg/mL Thermolysin.

4. Incubate reaction at 37 °C for 30 minutes.

5. Stop reaction by adding 100 µL of 20 mM 1, 10 Phenanthroline to the reaction.

Activate rhTrypsin 2 with activated rhEnterokinase.

1. Dilute activated rhEnterokinase to 0.4 µg/mL in Assay Buffer.

2. Dilute rhTrypsin 2 to 200 µg/mL in Assay Buffer.

3. Mix 25 µL of 0.4 µg/mL rhEnterokinase with 25 µL of 200 µg/mL rhTrypsin 2.

4. Incubate reaction at room temperature for 30 minutes.

Perform reaction.

1. Dilute activated rhTrypsin 2 to 0.05 µg/mL in Assay Buffer.

2. Dilute substrate to 20 µM in Assay Buffer.

3. In a plate load 50 µL of 0.05 μg/mL of rhTrypsin 2 and include a Substrate Blank of 50 µL Assay Buffer.

4. Start the reaction by adding 50 µL of 20 µM Substrate to wells.

5. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.

6. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

• rhTrypsin 2: 0.0025

• Substrate: 10 μM

Background: Trypsin 2/PRSS2

Human Trypsin 2, encoded by the PRSS2 gene, is also known as anionic Trypsin (1). Accounting for approximately 1/3 of the total trypsin content in the pancreatic juice, it is one of the trypsins produced in the pancreas. It consists of a signal peptide (residues 1‑15), a pro region (residues 16‑23), and a mature chain (residues 24‑247). Trypsin 2 is synthesized as a zymogen and secreted into the duodenal lumen, where it is activated by enteropeptidase (2). It plays a central role in digestion and activating other pro-enzymes. In human pancreatic diseases and chronic alcoholism, it is up-regulated (3).

• References:

1. Emi, M. et al. (1986) Gene. 41:305.

2. Halfon, S. et al. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) pp. 1483-1488, Academic Press, San Diego.

3. Rinderknecht, H. et al. (1979) Gut. 20:886.

• Entrez Gene IDs:

  5645 (Human); 22072 (Mouse); 25052 (Rat)

• Alternate Names:

  Anionic trypsinogen; EC 3.4.21; EC 3.4.21.4; MGC111183; MGC120174; MGC120175; protease serine 2 preproprotein; protease, serine, 2 (trypsin 2); protease, serine, 2, preproprotein; PRSS2; Serine protease 2; Tesp4; TRY2TRY8; TRY8; TRYP2; Trypsin 2; Trypsin II; trypsin-2; trypsinogen 2; Trypsinogen 20